Human iPSC model reveals a central role for NOX4 and oxidative stress in Duchenne cardiomyopathy.

Duchenne muscular dystrophy (DMD) is a progressive muscle disorder caused by mutations in the Dystrophin gene. Cardiomyopathy is a major cause of early death. We used DMD-patient-specific human induced pluripotent stem cells (hiPSCs) to model cardiomyopathic features and unravel novel pathologic insights. Cardiomyocytes (CMs) differentiated from DMD hiPSCs showed enhanced premature cell death due to significantly elevated intracellular reactive oxygen species (ROS) resulting from depolarized mitochondria and increased NADPH oxidase 4 (NOX4). CRISPR-Cas9 correction of Dystrophin restored normal ROS levels. ROS reduction by N-acetyl-L-cysteine (NAC), ataluren (PTC124), and idebenone improved hiPSC-CM survival. We show that oxidative stress in DMD hiPSC-CMs was counteracted by stimulating adenosine triphosphate (ATP) production. ATP can bind to NOX4 and partially inhibit the ROS production. Considering the complexity and the early cellular stress responses in DMD cardiomyopathy, we propose targeting ROS production and preventing detrimental effects of NOX4 on DMD CMs as promising therapeutic strategy.

Safety and efficacy of once-daily risdiplam in type 2 and non-ambulant type 3 spinal muscular atrophy (SUNFISH part 2): a phase 3, double-blind, randomised, placebo-controlled trial.

BACKGROUND: Risdiplam is an oral small molecule approved for the treatment of patients with spinal muscular atrophy, with approval for use in patients with type 2 and type 3 spinal muscular atrophy granted on the basis of unpublished data. The drug modifies pre-mRNA splicing of the SMN2 gene to increase production of functional SMN. We aimed to investigate the safety and efficacy of risdiplam in patients with type 2 or non-ambulant type 3 spinal muscular atrophy. METHODS: In this phase 3, randomised, double-blind, placebo-controlled study, patients aged 2-25 years with confirmed 5q autosomal recessive type 2 or type 3 spinal muscular atrophy were recruited from 42 hospitals in 14 countries across Europe, North America, South America, and Asia. Participants were eligible if they were non-ambulant, could sit independently, and had a score of at least 2 in entry item A of the Revised Upper Limb Module. Patients were stratified by age and randomly assigned (2:1) to receive either daily oral risdiplam, at a dose of 5·00 mg (for individuals weighing ≥20 kg) or 0·25 mg/kg (for individuals weighing <20 kg), or daily oral placebo (matched to risdiplam in colour and taste). Randomisation was conducted by permutated block randomisation with a computerised system run by an external party. Patients, investigators, and all individuals in direct contact with patients were masked to treatment assignment. The primary endpoint was the change from baseline in the 32-item Motor Function Measure total score at month 12. All individuals who were randomly assigned to risdiplam or placebo, and who did not meet the prespecified missing item criteria for exclusion, were included in the primary efficacy analysis. Individuals who received at least one dose of risdiplam or placebo were included in the safety analysis. SUNFISH is registered with ClinicalTrials.gov, NCT02908685. Recruitment is closed; the study is ongoing. FINDINGS: Between Oct 9, 2017, and Sept 4, 2018, 180 patients were randomly assigned to receive risdiplam (n=120) or placebo (n=60). For analysis of the primary endpoint, 115 patients from the risdiplam group and 59 patients from the placebo group were included. At month 12, the least squares mean change from baseline in 32-item Motor Function Measure was 1·36 (95% CI 0·61 to 2·11) in the risdiplam group and -0·19 (-1·22 to 0·84) in the placebo group, with a treatment difference of 1·55 (0·30 to 2·81, p=0·016) in favour of risdiplam. 120 patients who received risdiplam and 60 who received placebo were included in safety analyses. Adverse events that were reported in at least 5% more patients who received risdiplam than those who received placebo were pyrexia (25 [21%] of 120 patients who received risdiplam vs ten [17%] of 60 patients who received placebo), diarrhoea (20 [17%] vs five [8%]), rash (20 [17%] vs one [2%]), mouth and aphthous ulcers (eight [7%] vs 0), urinary tract infection (eight [7%] vs 0), and arthralgias (six [5%] vs 0). The incidence of serious adverse events was similar between treatment groups (24 [20%] of 120 patients in the risdiplam group; 11 [18%] of 60 patients in the placebo group), with the exception of pneumonia (nine [8%] in the risdiplam group; one [2%] in the placebo group). INTERPRETATION: Risdiplam resulted in a significant improvement in motor function compared with placebo in patients aged 2-25 years with type 2 or non-ambulant type 3 spinal muscular atrophy. Our exploratory subgroup analyses showed that motor function was generally improved in younger individuals and stabilised in older individuals, which requires confirmation in further studies. SUNFISH part 2 is ongoing and will provide additional evidence regarding the long-term safety and efficacy of risdiplam. FUNDING: F Hoffmann-La Roche.

The clinical, histologic, and genotypic spectrum of SEPN1-related myopathy: A case series.

OBJECTIVE: To clarify the prevalence, long-term natural history, and severity determinants of SEPN1-related myopathy (SEPN1-RM), we analyzed a large international case series. METHODS: Retrospective clinical, histologic, and genetic analysis of 132 pediatric and adult patients (2-58 years) followed up for several decades. RESULTS: The clinical phenotype was marked by severe axial muscle weakness, spinal rigidity, and scoliosis (86.1%, from 8.9 ± 4 years), with relatively preserved limb strength and previously unreported ophthalmoparesis in severe cases. All patients developed respiratory failure (from 10.1±6 years), 81.7% requiring ventilation while ambulant. Histopathologically, 79 muscle biopsies showed large variability, partly determined by site of biopsy and age. Multi-minicores were the most common lesion (59.5%), often associated with mild dystrophic features and occasionally with eosinophilic inclusions. Identification of 65 SEPN1 mutations, including 32 novel ones and the first pathogenic copy number variation, unveiled exon 1 as the main mutational hotspot and revealed the first genotype-phenotype correlations, bi-allelic null mutations being significantly associated with disease severity (p = 0.017). SEPN1-RM was more severe and progressive than previously thought, leading to loss of ambulation in 10% of cases, systematic functional decline from the end of the third decade, and reduced lifespan even in mild cases. The main prognosis determinants were scoliosis/respiratory management, SEPN1 mutations, and body mass abnormalities, which correlated with disease severity. We propose a set of severity criteria, provide quantitative data for outcome identification, and establish a need for age stratification. CONCLUSION: Our results inform clinical practice, improving diagnosis and management, and represent a major breakthrough for clinical trial readiness in this not so rare disease.

Novel defects in collagen XII and VI expand the mixed myopathy/Ehlers-Danlos syndrome spectrum and lead to variant-specific alterations in the extracellular matrix.

PURPOSE: To date, heterozygous or homozygous COL12A1 variants have been reported in 13 patients presenting with a clinical phenotype overlapping with collagen VI-related myopathies and Ehlers-Danlos syndrome (EDS). The small number of reported patients limits thorough investigation of this newly identified syndrome, currently coined as myopathic EDS. METHODS: DNA from 78 genetically unresolved patients fulfilling the clinical criteria for myopathic EDS was sequenced using a next-generation panel of COL12A1, COL6A1, COL6A2, and COL6A3. RESULTS: Among this cohort, we identified four pathogenic heterozygous in-frame exon skipping (∆) defects in COL12A1, clustering to the thrombospondin N-terminal region and the adjacent collagenous domain (Δ52, Δ53, Δ54, and Δ56 respectively), one heterozygous COL12A1 arginine-to-cysteine substitution of unclear significance (p.(Arg1863Cys)), and compound heterozygous pathogenic COL6A1 variants (c.[98-6G>A];[301C>T]) in one proband. Variant-specific intracellular accumulation of collagen XII chains, extracellular overmodification of the long isoform and near-absence of the short isoform of collagen XII, and extracellular decrease of decorin and tenascin-X were observed for the COL12A1 variants. In contrast, the COL6A1 variants abolished collagen VI and V deposition and increased tenascin-X levels. CONCLUSION: Our data further support the significant clinical overlap between myopathic EDS and collagen VI-related myopathies, and emphasize the variant-specific consequences of collagen XII defects.

Renal function in children and adolescents with Duchenne muscular dystrophy.

Improved life expectancy and the need for robust tools to monitor renal safety of emerging new therapies have fueled the interest in renal function in Duchenne muscular dystrophy (DMD) patients. We aimed to establish a methodology to accurately assess their renal function. Twenty DMD patients (5-22 years) were included in this prospective study. After obtaining medical history, all patients underwent a clinical examination, 24-hour ambulatory blood pressure monitoring, ultrasound of the kidneys, direct GFR measurement ((51)Cr-EDTA, mGFR), complete blood and urine analysis. Seventeen of 20 patients were treated with corticosteroids and 5/20 with angiotensin converting enzyme inhibitor (lisinopril). No patient suffered from urinary tract infections or other renal diseases. Hypertension (systolic or diastolic blood pressure >P95) was found in 9/20 patients (8/9 patients were on steroid treatment) and a non-dipping blood pressure profile in 13/20 subjects (10/13 patients were on steroid treatment). Urinary protein to creatinine ratio was elevated in 17/18 patients, whereas 24-hour urine protein excretion was normal in all subjects. Median interquartile range (IQR) mGFR was 130.4 (29.1) mL/min/1.73 m(2). Hyperfiltration (mGFR >150 mL/min/1.73 m(2)) was found in 5/20 patients. Inverse correlation between mGFR and age was observed (R(2) = 0.45, p = 0.001). Serum creatinine based estimated GFR (eGFR) equations overestimated mGFR up to 300%. eGFR based on cystatin C Filler equation was closest to the mGFR (median eGFR (IQR) of 129.5 (39.7) mL/min/1.73 m(2)). Our study demonstrates a high prevalence of hyperfiltration and hypertension in children and adolescents with DMD. Because the majority of hypertensive patients were under corticosteroid treatment, the iatrogenic cause of hypertension cannot be excluded. Serum or urine creatinine measurements are of no value to evaluate renal function in DMD patients due to the reduced skeletal muscle mass.

Homozygosity for aquaporin 7 G264V in three unrelated children with hyperglyceroluria and a mild platelet secretion defect.

PURPOSE: Aquaporin 7 (AQP7) belongs to the aquaglyceroporin family, which transports glycerol and water. AQP7-deficient mice develop obesity, insulin resistance, and hyperglyceroluria. However, AQP7's pathophysiologic role in humans is not yet known. METHODS: Three children with psychomotor retardation and hyperglyceroluria were screened for AQP7 mutations. The children were from unrelated families. Urine and plasma glycerol levels were measured using a three-step enzymatic approach. Platelet morphology and function were studied using electron microscopy, aggregations, and adenosine triphosphate (ATP) secretion tests. RESULTS: The index patients were homozygous for AQP7 G264V, which has previously been shown to inhibit transport of glycerol in Xenopus oocytes. We also detected a subclinical platelet secretion defect with reduced ATP secretion, and the absence of a secondary aggregation wave after epinephrine stimulation. Electron microscopy revealed round platelets with centrally located granules. Immunostaining showed AQP7 colocalization, with dense granules that seemed to be released after strong platelet activation. Healthy relatives of these patients, who were homozygous (not heterozygous) for G264V, also had hyperglyceroluria and platelet granule abnormalities. CONCLUSION: The discovery of an association between urine glycerol loss and a platelet secretion defect is a novel one, and our findings imply the involvement of AQPs in platelet secretion. Additional studies are needed to define whether AQP7 G264V is also a risk factor for mental disability.

The effect of Luque-Galveston fusion on curve, respiratory function and quality of life in Duchenne muscular dystrophy.

The aim of this retrospective study was to evaluate the long-term effect of the Luque-Galveston spinal fusion in Duchenne muscular dystrophy (DMD) patients. Twenty patients had undergone this operation at a mean age of 153 years (surgical group, A). The correction of their scoliosis amounted to +/- 55.8%, after an average follow-up period of 3 years. This is in accordance with the literature. The authors would therefore advise to perform spinal fusion in an early stage of the disease, once a rapid evolution of the scoliosis is seen. The decline of respiratory function slightly diminished after surgery, but not significantly. This means that no expectations should be made to improve respiratory function, as respiratory function decline continues relentlessly. Most authors agree with this statement. Patient satisfaction after surgery was relatively high, mainly because of an improved sitting balance, but only 60% of the questionnaires were available. Twenty-five other patients were not operated upon (non-surgical group, B). They had better results at ages 153 and 183, but this was mainly due to the fact that group B contained more benign cases according to the Oda classification.

Systemic administration of PRO051 in Duchenne's muscular dystrophy.

BACKGROUND: Local intramuscular administration of the antisense oligonucleotide PRO051 in patients with Duchenne's muscular dystrophy with relevant mutations was previously reported to induce the skipping of exon 51 during pre-messenger RNA splicing of the dystrophin gene and to facilitate new dystrophin expression in muscle-fiber membranes. The present phase 1-2a study aimed to assess the safety, pharmacokinetics, and molecular and clinical effects of systemically administered PRO051. METHODS: We administered weekly abdominal subcutaneous injections of PRO051 for 5 weeks in 12 patients, with each of four possible doses (0.5, 2.0, 4.0, and 6.0 mg per kilogram of body weight) given to 3 patients. Changes in RNA splicing and protein levels in the tibialis anterior muscle were assessed at two time points. All patients subsequently entered a 12-week open-label extension phase, during which they all received PRO051 at a dose of 6.0 mg per kilogram per week. Safety, pharmacokinetics, serum creatine kinase levels, and muscle strength and function were assessed. RESULTS: The most common adverse events were irritation at the administration site and, during the extension phase, mild and variable proteinuria and increased urinary α(1)-microglobulin levels; there were no serious adverse events. The mean terminal half-life of PRO051 in the circulation was 29 days. PRO051 induced detectable, specific exon-51 skipping at doses of 2.0 mg or more per kilogram. New dystrophin expression was observed between approximately 60% and 100% of muscle fibers in 10 of the 12 patients, as measured on post-treatment biopsy, which increased in a dose-dependent manner to up to 15.6% of the expression in healthy muscle. After the 12-week extension phase, there was a mean (±SD) improvement of 35.2±28.7 m (from the baseline of 384±121 m) on the 6-minute walk test. CONCLUSIONS: Systemically administered PRO051 showed dose-dependent molecular efficacy in patients with Duchenne's muscular dystrophy, with a modest improvement in the 6-minute walk test after 12 weeks of extended treatment. (Funded by Prosensa Therapeutics; Netherlands National Trial Register number, NTR1241.).

Predominant fiber atrophy and fiber type disproportion in early ullrich disease.

Ullrich disease (congenital muscular dystrophy type Ullrich, UCMD) is a severe congenital disorder of muscle caused by recessive and dominant mutations in the three genes that encode the alpha-chains of collagen type VI. Little is known about the early pathogenesis of this myopathy. The aim of this study was to investigate early histological changes in muscle of patients with molecularly confirmed UCMD. Muscle biopsies were analyzed from 8 UCMD patients ranging in age from 6 to 30 months. Type I fiber atrophy and predominance were seen early, together with a widening of the fiber diameter spectrum, whereas no dystrophic features were apparent. A subpopulation of more severely atrophic type I fibers was apparent subsequently, including one biopsy that fulfilled the formal diagnostic criteria of histopathological fiber type disproportion (FTD). Thus, early in the disease, UCMD presents as a non-dystrophic myopathy with predominant fiber atrophy. Collagen VI mutations also qualify as a cause of fiber type disproportion.

Increased Gs signalling in platelets and impaired collagen activation, due to a defect in the dystrophin gene, result in increased blood loss during spinal surgery.

Controversy exists regarding the cause of the significantly increased blood loss during spinal surgery in Duchenne muscular dystrophy (DMD) patients compared with similar surgery in other patients. DMD is caused by a mutation in the cytoskeletal dystrophin, which binds to extracellular matrix laminin and which has been described as a G-protein-coupled receptor. We hypothesized that disturbed cytoskeleton organization in DMD patients would alter Gs protein and collagen signalling in platelets, leading to dysfunctional platelets and a haemorrhagic tendency during surgery. In the present study, we found that platelets and skin fibroblasts, respectively, express the Dp71 and Dp116 dystrophin isoforms. Absent or decreased expression of these isoforms in DMD patients correlates with significant Gs alpha upregulation. Moreover, dysfunctional dystrophin in these cells is accompanied with increased Gs signalling and higher cAMP levels after Gs stimulation. Functional analysis showed that DMD platelets responded slower to collagen with an extensive shape change in the aggregometer and with a significantly reduced platelet adhesion to coated collagen under flow. The decreased collagen activation was shown to result from both Gs activation and cytoskeletal disruption and not from decreased expression of platelet membrane receptors or impaired von Willebrand factor (vWF) activity. In conclusion, DMD platelets have a disorganized cytoskeleton and manifest Gs hyperactivity and reduced platelet collagen reactivity. Their increased bleeding during surgery will, at least partly, result from the increased platelet Gs activity after the release of natural Gs agonists as prostacyclin during surgery and an ineffective reactivity to collagen.

Local dystrophin restoration with antisense oligonucleotide PRO051.

BACKGROUND: Duchenne's muscular dystrophy is associated with severe, progressive muscle weakness and typically leads to death between the ages of 20 and 35 years. By inducing specific exon skipping during messenger RNA (mRNA) splicing, antisense compounds were recently shown to correct the open reading frame of the DMD gene and thus to restore dystrophin expression in vitro and in animal models in vivo. We explored the safety, adverse-event profile, and local dystrophin-restoring effect of a single, intramuscular dose of an antisense oligonucleotide, PRO051, in patients with this disease. METHODS: Four patients, who were selected on the basis of their mutational status, muscle condition, and positive exon-skipping response to PRO051 in vitro, received a dose of 0.8 mg of PRO051 injected into the tibialis anterior muscle. A biopsy was performed 28 days later. Safety measures, composition of mRNA, and dystrophin expression were assessed. RESULTS: PRO051 injection was not associated with clinically apparent adverse events. Each patient showed specific skipping of exon 51 and sarcolemmal dystrophin in 64 to 97% of myofibers. The amount of dystrophin in total protein extracts ranged from 3 to 12% of that found in the control specimen and from 17 to 35% of that of the control specimen in the quantitative ratio of dystrophin to laminin alpha2. CONCLUSIONS: Intramuscular injection of antisense oligonucleotide PRO051 induced dystrophin synthesis in four patients with Duchenne's muscular dystrophy who had suitable mutations, suggesting that further studies might be feasible.

A mitochondrial tRNA aspartate mutation causing isolated mitochondrial myopathy.

Several mutations in mitochondrial transfer RNA (tRNA) genes can cause mitochondrial myopathy. We describe a young girl who presented with pronounced exercise intolerance. The anaerobic threshold and the maximal oxygen consumption were decreased. She had decreased complex I and IV enzyme activity and ragged red fibers on muscle biopsy. An A to G transition at nucleotide position 7526 in tRNA Aspartate (tRNA(Asp)) gene was heteroplasmic in several of the patient's tissues. We were unable to detect the mutation in muscle tissue from the patient's mother. This case adds a new genetic etiology for mitochondrial myopathy. It also illustrates for patients with combined deficiency of the complex I and IV enzyme activity the value of sequencing in the affected tissue muscle, and not only in blood, all mitochondrial tRNA genes including those not commonly affected, such as in this case mt tRNA(Asp).

Mutations of the selenoprotein N gene, which is implicated in rigid spine muscular dystrophy, cause the classical phenotype of multiminicore disease: reassessing the nosology of early-onset myopathies.

Multiminicore disease (MmD) is an autosomal recessive congenital myopathy characterized by the presence of multiple, short core lesions (known as "minicores") in most muscle fibers. MmD is a clinically heterogeneous condition, in which four subgroups have been distinguished. Homozygous RYR1 mutations have been recently identified in the moderate form of MmD with hand involvement. The genes responsible for the three other forms (including the most prevalent phenotype, termed the "classical" phenotype) remained, so far, unknown. To further characterize the genetic basis of MmD, we analyzed a series of 62 patients through a combined positional/candidate-gene approach. On the basis of clinical and morphological data, we suspected a relationship between classical MmD and the selenoprotein N gene (SEPN1), which is located on chromosome 1p36 (RSMD1 locus) and is responsible for the congenital muscular dystrophy with rigid spine syndrome (RSMD). A genomewide screening, followed by the analysis of 1p36 microsatellite markers in 27 informative families with MmD, demonstrated linkage to RSMD1 in eight families. All showed an axial myopathy with scoliosis and respiratory failure, consistent with the most severe end of the classical MmD spectrum; spinal rigidity was evident in some, but not all, patients. We excluded linkage to RSMD1 in 19 families with MmD, including 9 with classical MmD. Screening of SEPN1 in the 8 families that showed linkage and in 14 patients with classical sporadic disease disclosed 9 mutations affecting 17 patients (12 families); 6 were novel mutations, and 3 had been described in patients with RSMD. Analysis of three deltoid biopsy specimens from patients with typical RSMD revealed a wide myopathological variability, ranging from a dystrophic to a congenital myopathy pattern. A variable proportion of minicores was found in all the samples. The present study represents the first identification of a gene responsible for classical MmD, demonstrates its genetic heterogeneity, and reassesses the nosological boundaries between MmD and RSMD.